The development of a novel strategy to genetically modify neural stem/progenitor cells (NSCs) both in vitro and in vivo will have a significant impact on the development of NSCs-mediated cell therapy for neurological disorders as well as on the advance of basic developmental neurobiology. We recently have developed a packaging system for herpes simplex virus (HSV)-based amplicon vectors free from helper virus contamination or viral gene expression. Using this technology, we have demonstrated that genomic DNA inserts over 100 kilobases can be packaged into HSV virions, delivered intact, and expressed in cultured cells in vitro. By adding Epstein-Barr virus (EBV)-based replicon elements, HSV amplicons with genomic DNA inserts (HSV/EBV/genomic vectors) were shown to replicate and be maintained as extra chromosomal elements in infected cells, and express functional gene products for a long period of time. The unique properties of this vector system lead us to hypothesize that HSV/EBV/genomic vectors can be a platform technology to achieve some of the long-term goals toward the development of NSCs-mediated cell therapy, namely: 1) Complementing genetic defects of diseased NSCs in vitro and in vivo to treat genetic neurological disorders, 2) stable and cell type-specific transgene expression in neurons and glial cells differentiated from the transduced NSCs, and 3) genetic engineering of NSCs for controlled proliferation, migration, and differentiation in vivo after transplantation. To evaluate the feasibility of this approach, this project proposes to: 1) Verify episomal replication and maintenance of HSV/EBV/genomic vectors and evaluate furictional transgene expression from the vectors in dividing NSCs in culture, and 2) verify stable and cell-type-specific expression of genomic transgenes in terminally differentiated neurons and glial cells both in vitro and in vivo. We will examine five human genes, hypoxanthine-phosphoribosyltransferase, nestin, myelin basic protein, glial fibrillary acidic protein, and microtubule-associated protein tau as a housekeeping, NSC-specific, oligodendrocyte-specific, astrocyte-specific, and neuron-specific gene, respectively. These studies will provide further understanding of HSV/EBV/genomic vectors and confirm the advantages of genomic transgenes and EBV elements, which then should provide a strong basis for genetic engineering of NSCs using HSV/EBV/genomic vectors